Wa shington University 
Doctoral Dissertations 



The Toxic Property of Sulphur 



BY 



HARRY CURTIS YOUNG 




A Dissertation presented to the Faculty of Arts and 
Sciences in partial fulfilment of the requirements 
for the degree of Doctor of Philosophy, June, 1923 

Reprinted from Annals of the Missouri Botanical Garden 
November, 1922, Vol. IX, No. 4 



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Series V Number 39 



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University Studies 






V. 



/ 
^ THE TOXIC PROPERTY OF SULPHURS 

HAERY CURTIS YOUNG 
Research Fellow in the Henry Shmv School of Botany of Washington University^ 

Introduction 

Since the introduction of spraying for the control of parasitic 
fungi there has been developed a large number of fungicidal 
mixtures. Some have proved effective for the control of one 
organism and some for another, none of them having universal 
fungicidal value. Because of its abundance, low cost, and its 
effectiveness under certain conditions, sulphur has been employed 
in many of these mixtures. The fact that it has toxic or fun- 
gicidal properties has been conclusively demonstrated. In this 
work, an attempt has been made to determine if possible the 
exact nature of this fungicidal property, that is, to determine 
or evaluate the chemical compound or compounds in which this 
toxic property is resident, at the same time to relate this toxic 
property to conditions under which it may be consistently mani- 
fest, thus warranting its general use as a fungicide. 

The use of sulphur as a fungicide probably antedates that 
of all other substances. The chemical and physical properties 
of sulphur, especially its existence in so many forms, have led 
to its employment as a fungicide in a variety of ways. Regardless 
of the form in which it is employed, whether as a compound 
or as uncombined sulphur, there seem to be necessary certain 
chemical or physical changes before its toxic properties are ex- 
hibited. Toxicity has been attributed to many of the forms, 
for example, to such products of combined sulphur as various 
sulphides, thiosulphates, sulphur dioxide, sulphuric and sulphur- 
ous acids, and also to uncombined sulphur as flowers, or even as 
sulphur in a more finely divided state, that is, as colloidal sulphur. 
However, there seems to be no tangible evidence in the past 
work that toxic properties can be attributed directly to any one 
of these forms, the presence of which might thus determine its 
value as a fungicide. The exact state or states in which sulphur 
is toxic was left as a matter of considerable speculation. 

^ An investigation carried out at the Missouri Botanical Garden in the 
Graduate Laboratory of the Henry Shaw School of Botany of Washington 
University, and submitted as a thesis in partial fulfillment of the requirements 
for the degree of doctor of philosophy in the Henry Shaw School of Botany of 
Washington University. 

^ A fellowship established by the Crop Protection Institute for the investiga- 
tion of sulphur as a fungicide. 

(403) 



[Vol. 9 

404 annals of the missouri botanical garden 

History 

The generally employed sulphur sprays, namely, flowers of 
sulphur and the various sulphide compounds, have been only 
partially effective in controlling fungous diseases. It will not 
be necessary in this paper to go into a historical discussion of the 
effectiveness of these sprays, as such discussions are reported 
frequently by agricultural experiment stations and horticultural 
societies in bulletins and spray calendars and my own conception 
of the practical problems involved will be stated below. This 
work has to do largely with the fungicidal properties of sulphur. 

The toxicity of the flowers of sulphur has been attributed to 
several compounds, of which hydrogen sulphide, sulphur dioxide, 
sulphurous and sulphuric acids, and volatile sulphur are more 
often given. PoUacci ('07) believes that sulphur is transformed 
into sulphuretted hydrogen, the vapors of which have a very 
energetic action on the fungi. This view, however, has received 
but Httle support and has been proved untenable by Bourcart 
('13). He was unable to collect any of this gas on passing air 
from sulphur through solutions suitable for retaining the gas. 
Foreman ('10) could obtain no inhibition of germination with 
spores of Botrytis cinerea, using a saturated solution of sulphu- 
retted hydrogen. Similar results were obtained by Barker, Gim- 
ingham, and Wiltshire ('20). It is at present generally accepted 
that hydrogen sulphide is not a factor as a fungicidal property of 
sulphur. 

The view that the toxic action of sulphur is due to sulphur 
dioxide has received considerable support. Sostegni and Mori 
('90), Blodgett ('13), Butler ('17), and Kuhl ('21) conclude that 
the toxic property of sulphur is due to this gas. They believe 
that the gas is slowly produced by the oxidation of the sulphur. 
In the papers cited there is little substantiating experimental 
evidence, other than the fact that the toxic compound is volatile. 
Contrary views are held by Bourcart ('13) who states that ''Sul- 
phurous acid must not be dreamt of; 1/40,000 of this acid in 
the air would burn the leaves." In a series of experiments he 
could not collect any sulphur dioxide evolving from sulphur at 
temperatures up to 50° C. Barker, Gimingham, and Wiltshire 
('20) obtained good germination of spores of Nectria ditissima 
in a 1 : 100 solution of sulphur dioxide. Closed-ring experiments, 
however, gave limits of .005 per cent and .0005 per cent for the 
germination of spores of Sclerotinia jnictigena, Fusicladium den- 



1922] 

YOUNG THE TOXIC PROPERTY OF SULPHUR 405 

driticum, F. Pyrinum, Botrytis cinerea, and Nectria ditissima. 
They conclude that sulphur dioxide cannot be a factor. 

Marcille ('11) attributed the toxic property of sulphur to 
sulphur trioxide and sulphuric acid in the control of grape mil- 
dew. A similar conclusion was arrived at by Moissan ('04) who 
was able to obtain this gas from the spontaneous oxidation of 
different kinds of sulphur at ordinary temperatures. As far as 
the author is aware, these results have never been confirmed, and 
Bourcart ('13) and Barker, Gimingham, and Wiltshire ('20) 
proved on the contrary that sulphur trioxide and sulphuric acid 
do not contribute to the fungicidal property of sulphur. 

That sulphur is toxic because of its volatilization as such is 
probably the view most commonly held at the present time. 
The fact that spores are inhibited in germination when not in 
direct contact with the sulphur particle has been frequently dem- 
onstrated. Smith ('06), working with asparagus rust, con- 
cluded that sulphur acts by its fumes but that the sulphur must 
be uniformly distributed to be effective in controlling the disease. 
He found that the disease was best controlled in air pockets which 
aided in preventing a too rapid spreading and dilution of the 
fumes. Similar views are held by Mares and Mohr (see Bour- 
cart, '13), Bioletti ('07), Bourcart ('13), Barker, Gimingham, 
and Wiltshire ('20), Doran ('22), and others. 

The conditions under which sulphur is volatile or under which 
volatile substances are formed from sulphur have been inves- 
tigated by Marcille ('11), Bourcart ('13), Blodgett ('13), Kuhl 
('21), and Doran ('17, '22), with the following general con- 
clusions: (1) a certain temperature must be maintained, usually 
above 25° C; (2) oxygen is necessary; (3) sunlight is a possible 
factor; (4) the influence of the leaves and spores is considered 
by some a factor. These conclusions were arrived at by the use 
of flowers of sulphur. 

The toxicity of other forms, such as finely divided sulphur and 
the various sulphides, has been investigated by a number of 
workers. Doran ('22) found that Schloesing's precipitated sul- 
phur^ was more effective in killing spores of Venturia inaequalis 
than any of the finely divided sulphurs used. Atomic sulphur^ 
has been reported effective. 

^ Manufactured by Usines Schloesing Freres et Cie., of Marseille, France. 
'Prepared by the General Chemical Co., New York and Baltimore. 



[Vol. 9 
406 ANNALS OF THE MISSOURI BOTANICAL GARDEN 

Since the origination of lime sulphur as an orchard spray by 
M. F. Dusey of Fresno, California, in 1886, there has been a num- 
ber of studies made on its effectiveness as a spray and on its 
chemical composition. The first of these of importance was by 
Thatcher ('06). He found that lime sulphur contained for the 
most part calcium polysulphides, calcium thiosulphate, and small 
quantities of sulphites and sulphates. Haywood ('09), using the 
same methods, obtained similar results. When he dried the mix- 
ture the polysulphides disappeared and increasing amounts of 
precipitated sulphur were formed. He attributed the fungicidal 
value of lime sulphur particularly to the thiosulphates and pos- 
sibly to a combined or a summation of the toxic properties of all 
the compounds formed exclusive of sulphur. 

Van Slyke, Bosworth, and Hedges ('10) made some chemical 
determinations of lime sulphur when the ingredients were varied. 
They came to the conclusion that a mixture containing a high 
proportion of sulphur had the largest amount of calcium penta- 
sulphides and a greater fungicidal value. They proposed the 
following formula: 80 lbs. sulphur, 36 lbs. calcium oxide, and 50 
gallons water. This formula is the one in general use at the 
present time. Their chemical determinations gave about the 
same results as those obtained by Haywood ('09). Ruth ('13), 
in a study of lime sulphur and lead arsenate mixtures, found that 
no arsenic sulphide was formed. The proportion of thiosulphates 
and sulphites was increased in this mixture, and he attributed 
the increased effectiveness of the spray to the presence of ad- 
ditional quantities of these compounds. There was no experi- 
mental evidence for this, and his chemical determinations did 
not show the presence of any particular toxic compound. Harris 
('11) made chemical determinations of lime sulphur mixtures 
and found about the same amounts of sulphides, sulphites, etc., 
as Haywood. He also stated that filtering was unnecessary. Of- 
ficial methods for the determination of the compounds formed in 
lime sulphur are given by Roark ('20) and Winter ('20). 

The above studies on lime sulphur have had to do with freshly 
prepared mixtures. Vermorel and Dantony ('19) gave a number 
of reactions that probably took place in lime sulphur mixtures 
and the compounds formed when the mixture was aerated. 
They found that the polysulphides soon disappeared after the 
spray was applied. The calcium thiosulphates gradually decreased, 
and sulphites, sulphates, and free sulphur increased. Barker, 



1922] 

YOUNG THE TOXIC PROPERTY OF SULPHUR 407 

Gimingham, and Wiltshire ('20) concluded that calcium thio- 
sulphate, hydrogen sulphide, and sulphur dioxide were all slight- 
ly toxic but not sufficiently so to account for the fungicidal 
value of lime sulphur. The calcium pentasulphides were toxic, 
but since they disappeared in a few hours the lasting toxicity of 
lime sulphur could not be attributed to them. They concluded 
that the lasting toxic property must be due to precipitated sul- 
phur. Doran ('22) also found that the sulphides decomposed 
very rapidly, especially when dried slowly. 

Several other sulphide preparations have been employed as 
fungicides but have proved more or less ineffective as a lasting 
spray because their retention on the tree as sulphides is difficult 
to maintain. Their caustic nature frequently results in severe 
burning. 

In testing the toxicity of sulphur and its compounds consider- 
able confusion has developed owing to the variation in resistance 
of different species of fungi. Barker, Gimingham, and Wiltshire 
('20) found that germination of the spores of Sclerotinia jructi- 
gena and Phragmidium subcorticium were entirely inhibited in 
a suspension of flowers of sulphur in Van Tieghem cells. Ger- 
mination of Fusicladium dendriticum and Cladosporium fulvum 
were 50 per cent inhibited, while that of Nectria ditissima, Bo- 
trytis cinerea, and Verticillium sp. was not at all inhibited. When 
the flowers of sulphur was used Doran ('22) found that a much 
higher temperature was necessary for the killing of spores of 
Botrytis cinerea than for Venturia inaequalis and a higher tem- 
perature for the latter than for spores of Sclerotinia cinerea. 

Experimental 

Since most of the evidence listed in the foregoing references 
points to sulphur as being the toxic agent regardless of the sul- 
phur mixtures used, it was first thought important to study the 
influence of the sulphur particle and molecule on the germina- 
tion of spores. The Van Tieghem cell and the hanging-drop 
culture method, later slightly modified, were employed. The 
percentage of germination of the spores was used as an indication 
of toxicity. The organisms used were selected from the group of 
strict parasites most of which are of economic importance. It was 
also necessary to select those that sporulated readily. The fol- 
lowing forms were used: Colletotrichum Gossypii, Sclerotinia 
cinerea, Botrytis cinerea, Glomerella cingulata, Gloeosporium 



[Vol. 9 
408 ANNALS OF THE MISSOURI BOTANICAL GARDEN 

venetum, Macrosporium sarcinaeforme, Phomopsis Sojae, and 
Actinomyces Scabies. These organisms were grown on potato 
agar prepared according to the method of Duggar, Severy, and 
Schmitz ('17), and spores were taken from cultures 10-15 days 
old. 

The culture solution used in the hanging drops and in which 
the sulphur particles were suspended was a slightly buffered mix- 
ture containing mannite, phosphoric acid, and sodium hydroxide. 
The solution was prepared according to the method of Karrer 
and Webb ('20), as follows: Stock solutions of M/5 mannite in 
M/10 phosphoric acid and M/5 mannite in M/5 sodium hy- 
droxide were made. Equal quantities of the M/5 mannite- 
M/10 phosphoric acid were placed in each of 10 flasks and suc- 
cessively increasing proportions of M/5 mannite-M/5 sodium 
hydroxide were added. The flasks were plugged with cotton, 
sterilized at 15 lbs. pressure for 15 minutes, and allowed to stand 
for a few hours. Titrations made by the coloriraetric method 
(Clark, '20) showed the mixtures to have the following range of 
hydrogen-ion concentrations: Ph 1.6, 2,4, 3.2, 4.2, 5.2, 5.8, 6.4, 6.8, 
7.4, 8.4. 

EXPERIMENT I. TOXICITY OF THE FLOWERS OF SULPHUR 

Since sulphur in the form of flowers is insoluble in any solu- 
tion that can be used for the growing of fungi, it was necessary 
to test its toxicity in the form of suspensions. Twenty test-tubes 
were provided with pipettes that extended through the cork stop- 
pers and to the bottoms of the tubes. By this means drops could 
be transferred readily to the hanging-drop cells. These test- 
tubes constituted a duplicate series of 10 each, and 5 cc. of each 
of the slightly buffered solutions were added to the tubes so that 
each tube represented a particular hydrogen-ion concentration. 
To one series .5 gm. of flowers of sulphur was added to each tube. 

The technique of the planting of the hanging-drop cultures 
was essentially the same as that used by Webb ('21) in his ger- 
mination studies, and was as follows: Ground glass rings were 
cemented to glass slides by means of parawax and petrolatum. 
Two of these rings were placed on each slide, and 20 slides con- 
stituted a series for each organism. This gave duplicate cultures 
for each hydrogen-ion concentration. A few drops of the sul- 
phur suspension to be tested for its toxicity were placed in the 
bottom of the two cells. Another drop was placed on a clean 



1922] 

YOUNG THE TOXIC PROPERTY OF SULPHUR 409 

sterile glass slide. A loop-full of spores was placed in this drop 
and the spores evenly distributed throughout the drop. By- 
means of a small sterile glass rod a small portion of this drop was 
transferred to a clean sterile cover glass and the latter inverted 
over the glass cell. The cell was made air-tight by sealing with 
petrolatum. In like manner cultures were made representing 
each hydrogen-ion concentration both with and without sulphur. 
The series of hanging-drop cultures were then kept at room tem- 
perature. Examinations were made at the end of 16 and 24 hours. 

After examining some of the preliminary cultures it was found 
that considerable irregularity in germination existed. Some types 
of spores would remain on the surface of the drops and often 
would not be in close proximity to the sulphur. Other types 
of spores were found to be in the center of the drops with the 
sulphur particles. Different-sized drops would often result in 
irregular germination in the control cultures. With some or- 
ganisms the number of spores in the drop influenced the rate of 
germination. To eliminate such chance for error a definite spore 
suspension was made and the drop on the cover glass was spread 
over a much larger surface, giving it more the nature of a smear. 
In this way a more even distribution of both the spores and the 
sulphur particles was obtained. The results of the experiment 
are recorded in table i and figs. 1-4. 

Sulphur in this form was found to be directly toxic to only 
two of the organisms used. In the other forms the spores were 
not only not inhibited from germinating but the germ tubes 
grew normally when in direct contact with the sulphur particles. 
It can only be concluded from these results that if the flowers of 
sulphur has a general fungicidal value it must be due to some 
change in the form of sulphur and that this change takes place 
under different conditions than were obtained in closed-ring Van 
Tieghem cells. Within the usual range for germination the hy- 
drogen-ion concentration influenced the results but slightly. 

EXPERIMENT 2. FINELY GROUND FLOWERS OP SULPHUR 

Since the ordinary flowers of sulphur was toxic to two of the 
organisms, it was concluded that there was a toxic property pres- 
ent but in a very dilute form. If physical conditions influenced 
the production of this property it was thought that possibly a 
finely ground product might be more effective. To obtain sulphur 
in this state an electrically driven excentric mortar, as used for 



[Vol. 9 
410 ANNALS OF THE MISSOURI BOTANICAL GARDEN 

crushing yeast cells, was employed. Eight gms, of flowers of sul- 
phur were mixed with 3 gms. of diatomaceous earth (Kieselguhr), 
and the mixture ground for 14 hours. One-half gm. of this mix- 
ture was added to each test-tube containing the slightly buffered 
solution of the different hydrogen-ion concentrations and the 
toxicity determined as before. An attempt was made to grind 
the sulphur without the diatomaceous earth but the sulphur had 
a tendency to cake and did not grind well. Other substances are 
being tried with the hope of eliminating diatomaceous earth. Re- 
sults of this experiment are given in table i, figs. 1-4. 

Sulphur in this state was found to be more toxic than the 
flowers of sulphur unground. A more marked influence of the 
hydrogen-ion concentration was noted, the range showing the 
greatest toxicity being between Ph 4.2 and 5.4. The increased 
toxicity at this point is attributed to one of 2 possibilities: first, 
the spores may be less resistant at this point, or second, the toxic 
form or conditions of sulphur may have been produced in greater 
amounts at this range. At any rate the hydrogen-ion concentra- 
tion and the fineness of the particle contributed to the in- 
creased toxicity of the sulphur. The fineness of the particle 
did not seem to be the direct cause, as germ tubes grew normally 
after the initial retardation, even though they were directly in 
contact with the sulphur particles. 

EXPERIMENT 3. COLLOIDAL SULPHUR 

Sulphur readily assumes the colloidal state. The element sul- 
phur has been known since the beginning of history, and records 
show that colloidal sulphur was prepared and studied as early 
as the seventeenth century. "Lac Sulfurus," a colloidal form of 
sulphur, was prepared in 1765 by Stahl (1766) and was used at 
that time for medicinal purposes. Fourcroy (1790), Berthollet 
(1798), Berzelius (1808), and Magnus (1827) were early con- 
tributors to the study of colloidal sulphur. Present-day methods 
for the preparation of colloidal sulphur are found in papers by 
Svedberg ('09), Himmelbauer ('09), Raffo ('08, '11), Oden ('13), 
v. Weimarn and Molyschew ('11), Kelber ('12), and others. 

Colloidal sulphur exists in two forms, depending upon the 
degree of hydration. The form having a very high degree 
of hydration will be discussed in this paper as the hydro- 
philic colloidal sulphur and is identical with the product prepared 
by Raffo and Mancini ('11) and Oden ('13) and called "soluble 



YOUNG THE TOXIC PROPERTY OF SULPHUR 411 

colloidal sulphur." The other form of colloidal sulphur is that 
first prepared by v. Weimarn and Molyschew (11). This last 
has a very low degree of hydration and will be designated in 
this paper as hydrophobic colloidal sulphur. A more detailed 
description of these forms will be given in a subsequent section. 

The hydrophilic colloidal sulphur was prepared according to 
the methods of Raffo and Mancini ('11) and Oden ('13) with 
certain modifications. Fifty gms. of pure crystalline sodium thio- 
sulphate were dissolved in 30 cc. of distilled water; 70 gms. of 
concentrated sulphuric acid, sp. gr. 1.84, arsenic free, were 
weighed into a glass cylinder of 300 cc. capacity. The cylinder 
was placed in a vessel of cold water and the saturated solution of 
sodium thiosulphate added very slowly with occasional stirring. 
The mixture was then allowed to cool and 30 cc. of distilled water 
added. It was then placed on the water bath and warmed at 
80° C. for 10 minutes, and filtered through glass wool to re- 
move insoluble sulphur. The filtrate was cooled and allowed 
to stand for 12 hours. It was again warmed, filtered through 
glass wool, and the filtrate cooled. This warming, filtering, and 
cooling was repeated until no more insoluble sulphur came down. 
The final filtrate was a slightly turbid yellowish solution. This 
was centrifuged for 30 minutes at 1500 revolutions per minute. 
A portion of the colloidal sulphur was thrown out of solution. 
The supernatant liquid was a clear yellowish solution and was 
saved for further purification. The residue was washed in cold 
distilled water and again centrifuged for the same length of time 
and at the same speed. The supernatant liquid was again yel- 
lowish and was saved. The washing and centrifuging of the res- 
idual colloidal sulphur were repeated until the residue peptized 
in water gave a reaction of Ph 4.2. This colloidal suspension was 
faintly yellow and upon standing 1 week some of the particles 
settled out, the solution retaining its faint yellow color. Upon 
drying and weighing, the suspension gave a percentage of sulphur 
of 3.4. 

The supernatant liquids collected from the above were treated 
with a concentrated solution of sodium chloride, whereby the 
yellowish colloidal sulphur was coagulated. The sodium chloride 
was added until no more coagulum seemed to form. The coag- 
ulum was easily centrifuged out and repeptized in 10 cc. of dis- 
tilled water. The color of this solution was a deeper yellow and 
only very slightly turbid. This colloidal suspension gave a reaction 



[Vol. 9 
412 ANNALS OF THE MISSOURI BOTANICAL GARDEN 

of Ph 4.2 and did not settle out on standing for 2 months. On 
drying and weighing, the solution was found to contain 1.6 per 
cent sulphur. This latter preparation was a typical hydrophilic 
colloidal sulphur and was more nearly a true "soluble" sulphur 
than the product obtained from the method of Raffo and Man- 
cini ('11). The first preparation was a mixture of hydrophilic 
and hydrophobic colloidal sulphur. Oden ('13), in a detailed 
study of this type of colloidal sulphur mixtures, found them to 
contain particles of different sizes ranging from the molecular 
to particles easily discernible under the lov/ power of the micros- 
cope. He was able to obtain suspensions with particles varying 
from the smallest to the largest by fractional coagulation with 
sodium chloride. Particles of larger size were more easily coagu- 
lated than the smaller ones. In colloidal sulphur suspensions 
of this kind the particles have a tendency to collect themselves 
into groups, forming larger particles which settle out rapidly. 
The smaller the particles the slower this takes place and in hy- 
drophilic colloidal sulphur suspensions only a small amount of 
settling out can be noted after several months. 

The chemical reactions involved in the formation of colloidal 
sulphur prepared by this method is given by Oden as follows : 

NasSzOa + H2SO4 = Na2S04 + H2S2O3 
H2S2O3 -= SO2 + H2O + S 
2 H2S2O3 == 2H2S + 2SO2 
2 H2S + SO2 = H2O + S 

3H2S2O3 = 3H2O + H2SO4 + s + s 

Further chemical reactions will be given in a subsequent section 
of this paper. 

The method for the preparation of hydrophilic colloidal sul- 
phur was later varied in accordance with the method used by 
Freundlich and Scholz ('22). After the filtration through glass 
wool concentrated sodium chloride was added and the mixture 
centrifuged. The coagulum was then peptized with 100 cc. 
of distilled water and the insoluble sulphur centrifuged out. 
The peptized sulphur solution was treated 3 times with 25 
ec. of saturated sodium chloride and finally peptized in 100 cc. 
of distilled water. 

Another method for the preparation of hydrophilic colloidal 
sulphur was that first used by Selmi ('52) and was as follows. Sul- 
phur dioxide was passed into distilled water until a saturated 



1922] 

YOUNG THE TOXIC PROPERTY OF SULPHUR 413 

solution was formed. Hydrogen sulphide was then passed into 
the sulphurous acid solution, care being taken not to have an 
excess of the hydrogen sulphide, as it precipitates the hydrophilic 
colloidal sulphur forming the hydrophobic colloid. The solu- 
tion was then centrifuged to remove the larger particles and the 
supernatant liquid coagulated with sodium chloride. The coag- 
ulum was then peptized in water as before. 

The hydrophobic colloidal sulphur can be prepared in a num- 
ber of ways. It is the "milk of sulphur" formed when sulphur 
is precipitated out of solution. It was prepared in this work by 
the method used by v. Weimarn and Molyschew ('11) which 
was as follows: Sulphur was recrystallized in toluol and the 
toluol evaporated off at 60-70° C. Five-tenths gm. of this was 
heated with 125 cc. of fresh distilled absolute alcohol in a reflux 
condenser for 60 minutes. Seven cc. of this hot solution were 
poured into 293 cc. of distilled water at room temperature. The 
suspension prepared in this way was white and turbid. This was 
centrifuged and resuspended in water. The sulphur particles 
settled out of this suspension in a comparatively short time. 

In determining the toxicity of these forms of colloidal sulphur 
the same method was used as in the preceding tests. With the 
hydrophilic colloidal sulphur, however, it was necessary to make 
a much weaker suspension. The stock colloidal suspensions con- 
tained about 1.5 per cent sulphur. Five cc. of this stock sus- 
pension were diluted to 25 cc. with distilled water, and then 1 
cc. of this was added to each of the hydrogen-ion concentrations. 
This gave a further dilution of 1 : 5 and resulted in a very weak 
suspension of colloidal sulphur. After a preliminary test, how- 
ever, the hydrophobic colloidal sulphurs were not diluted with 
water, and 1 cc. of the stock suspension was added directly to 
the culture solutions. The organisms used and the results are 
recorded in table i and figs. 1-4. 

With the 6 organisms used in this experiment hydrophilic 
colloidal sulphur was found to be extremely toxic in the very 
dilute suspensions used. Only 2 of the organisms, namely, Bo- 
trytis cinerea and Macrosporium sarcinaeforme, showed a slight 
resistance to this suspension. In stronger suspensions germina- 
tion was entirely inhibited with all the organisms used. On the 
other hand, hydrophobic colloidal sulphur was only slightly toxic 
and comparable to ground flowers of sulphur. The results in- 
dicate that sulphur is most toxic in a very finely divided state 



414 



ANNALS OF THE MISSOURI BOTANICAL GARDEN 



[Vol. 9 



such as is found in the hydrophilic colloidal sulphur. The in- 
fluence of the hydrogen-ion concentration was very striking, es- 
pecially with this latter form of sulphur. Upon examination 
of the culture tubes containing the hydrophilic colloidal sulphur 
it was found that settling out was rapidly increased as the Ph 
incr8a>3sd beyond Ph 5,4. 
yR7 




\ 


riannik-tlFO^NaOtl 


\\ 


Doffered HiKture 


\\ 


\ s 


>> 


\ 


\\ 








\\ \ ^ 


;• 


\\ \\ 


,* 





/^^ 



Fig. 1. Germination of spores of Botrytis cinerea in hanging-drop cultures: 

toxic action of flowers of sulphur ; of hydrophobic colloidal sulphur 

— ' ; of hydrophilic colloidal sulphur ; check, without sulphur 



EXPERIMENT 4. THE TOXICITY OF LIME SULPHUR 

The compounds formed in lime sulphur mixtures have been 
fairly well determined by Ha3rwood, Van Slyke, and others. The 
reactions that take place when sulphur and calcium oxide are 
boiled together are about as follows: 

3Ca(OH)2 + 12S = CaS203 + 2CaS5 + SH^O 
or 3Ca(OH)2 + 88== CaS203 + CaSs + 3H2O 

These reactions are influenced, of course, by the initial ratio of 
the ingredients. Varying amounts of CaSs, CaS4, CaSs, CaS203 
and CaSOs are formed depending upon this ratio. When lime 
sulphur is prepared according to the Van Slyke method, that is, 



1922] 

YOUNG — THE TOXIC PROPERTY OF SULPHUR 415 

boiling together 80 lbs. of sulphur, 36 lbs. of lime, and 50 gal- 
lons of water, the first reaction is the more probable one. When 
prepared in this way the mixture has about the following com- 
position: sulphur as sulphides (largely pentasulphides), 80.7 
per cent, as thiosulphates, 19 per cent, as sulphites and sulphates, 
0.0? per cent. 

Lime sulphur mixtures are extremely alkaline and their initial 
eflSciency as a fungicide may be due partly to this causticity, that 
is, to the free hydroxyl ions. An experiment was performed to 
determine how long this causticity remained when the spray 



riamde n^PQ^-NaOM- 
Buffizrtd riixture 




Fig. 2. Germination of spores of Colletotrichum Gossypii in hanging-drop 

cultures: toxic action of flowers of sulphur ; of hydrophobic colloidal 

sulphur ; of hydrophilic colloidal sulphur ; cheek, without 

sulphur . 

was applied, and to ascertain, if possible, whether this factor was 
the principal one in giving lime sulphur its prolonged effective- 
ness as a fungicide. For this purpose lime sulphur was prepared 
according to the formula given by Van Slyke, and 1 part of the 
lime sulphur diluted with 6 parts of water. This is a little 
stronger than the concentration used as a dormant spray. 
Twelve large moist chambers were sprayed with this mixture and 
kept under the following conditions: Four were exposed to dry 
air; a second set of 4 was placed under slightly humid condi- 
tions, and a third set of 4 in a saturated condition. After 2 hours 



416 



ANNALS OF THE MISSOURI BOTANICAL GARDEN 



[Vol. 9 



the lime sulphur was washed from 1 of the exposed glass dishes 
and the reaction determined. It was found to have changed 
from an initial reaction of beyond the alkaline Ph range of in- 
dicators available (Ph 10.0) to Ph 6.4. Likewise, at the end of 
the same length of time the mixture was washed from one of the 
vessels in the second set and tested for its reaction. The reaction 
in this case remained beyond Ph 10. 

At the end of 6 hours the reactions were again determined. 
The wash from the first set remained the same. In the second 
set the reaction had changed to Ph 7.4 and in the third set it was 
still beyond Ph 10. At the end of 24 hours a third set of readings 



do 



.B 



^60 

o 

I 



or 



^ 



Mannite -MsFV^-NaOfi- 
Dufkred Hixture 




/ \ \\ 



■•■■■•V 



fhZ 3 4- 6.6 7 6 
Fig. 3. Germination of spores of Gloeosporium venetum in hanging-drop 
cultures: toxic action of flowers of sulphur ; of hydrophobic col- 
loidal sulphur ; of hydrophilic colloidal sulphur ; check, 

without sulphur . 

was made. All gave the same reaction, Ph 6.4. The mixture 
placed under the third condition did not dry out, but changed 
in reaction to the same point as the others. It would appear 
from these results that the lasting action of lime sulphur is not 
due to its causticity. 

At this point it was thought advisable to make some chemical 
determinations of the exposed or changed lime sulphur. Using 
the same method as that listed by the Association of Official 
Agricultural Chemists ('20) it was found that polysulphides were 
absent. The percentage of thiosulphates as determined by the 



1922] 



YOUNG THE TOXIC PROPERTY OF SULPHUR 



417 



Shaffer and Hartman method ('21) was 1.4. Sulphides were 
found to be approximately 0.1 per cent. Precipitated sul- 
phur as determined by the carbon bisulphide method gave 
a percentage of 2.8. We have present then in the changed lime 
sulphur, precipitated sulphur, calcium thiosulphate, calcium sul- 
phite, and calcium sulphate. 

The toxicity of these individual compounds was next deter- 
mined. Fifty cc. of 1:6 lime sulphur solution were set aside in a 
large open vessel. After 36 hours the reaction had changed to Ph 
6.4. The solution was then removed and the vessel washed with 



nannite-M^FO^-NaOti 
Buffered f^ixture 




PtiZ 3 ^ J ^ J ^ 
Fig. 4. Germination of spores of Macrosporium sarcinae forme in hanging- 
drop cultures: toxic action of flowers of sulphur ■ ; of hydrophobic col- 
loidal sulphur . ; of hydrophilic colloidal sulphur ; check, 

without sulphur . 

sufficient distilled water to make the total quantity up to the 
original 50 cc. This mixture was then centrifuged until the 
supernatant liquid was clear. A test was then made for calcium 
thiosulphate in the supernatant liquid. The percentage was 1.4. 
The part thrown out of the solution by the centrifuge was again 
washed in cold water and again centrifuged. The washing re- 
moved any soluble compound that might have been present. This 
washed substance was then suspended in 50 cc. of distilled water. 
The compounds contained in this suspension were the insoluble 



[Vol. 9 
418 ANNALS OF THE MISSOURI BOTANICAL GARDEN 

compounds that were formed in the changed lime sulphur, such 
as the sulphites and sulphates and the precipitated sulphur. The 
toxicity of these compounds was determined in the same way as 
in the preceding experiments. 

The calcium thiosulphate solution did not inhibit germination 
of any of the organisms used. Similar results were obtained by 
Armstrong ('21) in his studies on sulphur nutrition of the fungi. 
Accordingly, calcium thiosulphate cannot be a factor, even at 
this high concentration, in the fungicidal value of lime sulphur. 

One cc. of the precipitated sulphur suspension was placed in 
each of the tubes containing the slightly buffered solution. This 
made a further dilution of 1:5, making this final suspension 
equal to that of the original changed lime sulphur, that is, 1:6. 
The toxicity was determined in the same way as in the preceding 
tests. The results are given in table i. 

The results were very similar to those obtained with colloidal 
sulphur. The hydrogen-ion concentration influenced the tox- 
icity in the same general way. To make sure that this toxicity 
was not due to the sulphites and sulphates the sulphur was coag- 
ulated out as in the case of colloidal sulphur and the toxicity 
again determined. The results were the same. A further test 
was made, using a 0.1 per cent solution of calcium sulphite, but 
no toxicity resulted. An attempt was next made to try to further 
purify the sulphur suspension by fractional centrifugation, in 
which the centrifuge was run very slowly, thus throwing out the 
sulphur and not the insoluble calcium sulphites. By repeating 
the centrifuging 5 or 6 times a sulphur suspension was obtained 
which when dried was completely soluble in carbon bisulphide. 
The results with reference to toxicity were the same as those 
cited above. 

It must be concluded from these results that the lasting fun- 
gicidal value of lime sulphur is due almost entirely to the pre- 
cipitated sulphur, directly or indirectly, and not to the calcium 
thiosulphate and the insoluble sulphites. The precipitated sul- 
phur formed in the changed lime sulphur is not in as finely 
divided state as the soluble colloidal sulphur prepared by the 
above methods, as was shown by the slow speed with which it 
could be thrown out of suspension. However, its toxicity was 
slightly greater than that of the hydrophobic colloidal sulphur 
in the same concentration. 



1922] 



YOUNG THE TOXIC PROPERTY OF SULPHUR 

TABLE I 
PERCENTAGE OF GERMINATION* 



419 



Organism 



Botrytis cinerea. 



Colletotrichum 
Gossypii 



Form of Sulphur 



1.62.43.24.25.45.86.46.87.48.4 



Without sulphur 

Flowers of sulphur 

Ground flowers of sulphur 
Hydrophilic colloidal 

sulphur ._ 

Hydrophobic colloidal 

sulphur 

Precipitated lime sulphur 



Without sulphur 

Flowers of sulphur 

Ground flowers of sulphur 
Hydrophilic colloidal 

sulphur 

Hydrophobic colloidal 

sulphur 

Precipitated lime sulphur 



Sclerotinia 
cinerea... 



Without sulphur 

Flowers of sulphur 

Hydrophilic colloidal 
sulphur 



Gloeosporium 
venetum 



Without sulphur 

Flowers of sulphur 

Ground flowers of sulphur 
Hydrophilic colloidal 
sulphur 



Macrosporium 
sarcinaeforme . 



Phomopsis Sojae. 



Hydrogen-ion concentration (Ph) 



Without sulphur 

Flowers of sulphur 

Ground f owers of sulphur 
Hydrophilic colloidal 
sulphur 



Without sulphur 

Flowers of sulphur.. 



22 



18 
21 



20 



40 
41 
40 

38 

26 




* Average of 6-12 replications conducted at 3 different times. 

EXPERIMENT 5. THE TOXICITY OF THE VOLATILE 
PRODUCTS OF SULPHUR 

The results of the foregoing experiments indicate that sulphur 
is most toxic when it is in a finely divided state, this toxicity in- 
creasing in proportion to the fineness of the particle, hydrophilic 
colloidal sulphur exhibiting the highest degree of toxicity. The 
prevailing supposition that sulphur is only toxic when in a vol- 
atile state might be justified by the assumption that finely di- 



420 



ANNALS OF THE MISSOURI BOTANICAL GARDEN 



[Vol. 9 



vided sulphur yields a volatile product more readily. On the other 
hand, the peculiar relation of this toxicity to a definite range of 
hydrogen-ion concentration points rather towards the probability 
that sulphur is toxic because of a compound produced that may 
be volatile, the production of this compound being affected direct- 
ly by the reaction. It does not seem probable that the colloidal 
sulphur particle as such could be rendered non-toxic by so slight 
a change in reaction as has been shown to govern its fungicidal 
property. 

To determine these points a series of experiments was arranged, 
using flowers of sulphur, hydrophilic and hydrophobic ' colloidal 
sulphur. The organisms used were Botrytis cinerea, Colleto- 
trichum Gossypii, and Sclerotinia cinerea. The method of proce- 
dure was the same as in the preceding experiments, with the fol- 
lowing modifications: The spores were placed in drops of the 
slightly buffered solution without sulphur. The sulphur suspen- 
sions were placed only at the bottom of the cells. In this way the 
spores were not in direct contact with the sulphur, the distance 
between culture drop and cell liquid being the height of the cell, 
which was 8 mm. The cultures were incubated at 22° C. The 
results are given in table ii. 

TABLE II 

PERCENTAGE OF GERMINATION 



Organism 



Form of Sulphur 



Hydrogen-ion concentration (Ph) 



1.62.4 



3.2 



4.2 



5.45.8 



6.46.8 



7.48.4 



Botrytis cinerea. 



Without sulphur 

Flowers of sulphur 

Hydrophilic colloidal 

sulphur 

Hydrophobic colloidal 

sulphur 



10 



Colletotrichum 
Gossypii 



Without sulphur 

Flowers of sulphur 

Hydrophilic colloidal 

sulphur 

Hydrophobic colloidal 

sulphur 



Sclerotinia cinerea . 



Without sulphur 

Flowers of sulphur 

Hydrophilic colloidal 

sulphur 

Hydrophobic colloidal 

sulphur 



65 



^Precipitated sulphur. 



1922] 

Y0UNC3 THE TOXIC PROPERTY OF SULPHUR 421 

The results in this table are very similar to those recorded in 
table I except that the flowers of sulphur exhibited no toxic ac- 
tion even to Sclerotinia cinerea and the hydrophobic colloidal sul- 
phur was only slightly toxic with Botrytis cinerea and Colleto- 
trichum Gossypii. The hydrophilic colloidal sulphur exhibited 
the usual degree of toxicity, regardless of the fact that it was 
a considerable distance from the spore. Toxicity was greatest 
in all cases at Ph 4.0-5.5, as in the previous tests. 

Having determined that the toxic substance is volatile, it was 
thought necessary at this point to eliminate, if possible, hydrogen 
sulphide, sulphur dioxide, and sulphur trioxide, as factors. For 
these tests Sclerotinia cinerea was selected because it has proved 
to be quite sensitive to the toxic action of sulphur. Spores were 
placed over a saturated solution of hydrogen sulphide in a Van 
Tieghem cell and the cultures were incubated at 22° C. for 24 
hours. Germination was not inhibited. The experiment was re- 
peated with Colletotrichum Gossypii and Botrytis cinerea with 
similar results. 

No toxicity could be noted with sulphur dioxide in a concentra- 
tion sufficient to kill when converted into hydrophilic colloidal 
sulphur by the addition of hydrogen sulphide. 

Sulphuric acid inhibited growth only because of its acidity, ac- 
cordingly, in proportion to acidity. Positive tests for sulphur 
dioxide and trioxide could not be obtained in aerated sulphur 
suspensions that were toxic to Sclerotinia cinerea. These com- 
pounds, therefore, do not contribute to the toxic properties of 
sulphur. 

EXPERIMENT 6. THE INFLUENCE OF O2 ON THE TOXICITY 
OF SULPHUR 

In all of the foregoing tests the only oxygen available was 
that present in the air enclosed in the closed-ring cells. An ex- 
periment was conducted to determine the effect of oxygen on in- 
creasing the toxicity of flowers and precipitated sulphur. Finely 
ground flowers of sulphur and hydrophobic colloidal sulphur 
were placed in the slightly buffered mixtures in the same concen- 
tration as in Experiments 1 and 2. The Van Tieghem cells were 
placed in Petri dishes, the bottoms of which were lined with filter- 
paper in which holes somewhat larger than the glass rings were 
cut, so that the cells might rest on the bottoms of the Petri 
dishes. A large drop of the sulphur suspension was placed at 



422 



[Vol. 9 



ANNALS OF THE MISSOURI BOTANICAL GARDEN 



the bottom of the ring. The filter-paper was saturated with 
water. Spores of Sderotinia cinerea were placed in a drop of the 
culture medium without sulphur, on a cover slip which was in- 
verted over the cell. The cells were not sealed at the top or 
bottom. 

In the same Petri dish sealed cells were prepared. This was 
done for each hydrogen-ion concentration. The Petri dishes 
containing the cultures were arranged in a moist chamber through 
which air was passed. This experiment was conducted at room 
temperature and the percentage of germination noted after 18 
hours. The results are given in table iii. 



TABLE III 

PERCENTAGE OP GERMINATION* 
SCLEROTINIA CINEREA 





Ground flowers of sulphur 


Hydrophobic colloidal sulphur 


Ph 












-O2 


+ O2 


-O2 


+ O2 


2.4 


40 


38 


34 


24 


3.2 


64 


49 


17 


10 


4.2 


68 


31 


8 





5.4 


65 


24 


11 





5.8 


62 


46 


54 


32 


6.4 


58 


49 


48 


44 



*Average of triplicate cultures. 

Another experiment was conducted, in which a weak suspension 
of hydrophobic colloidal sulphur which did not inhibit the ger- 
mination of spores of Colletotrichum Gossypii at any hydrogen- 
ion concentration was aerated for 24 hours. Air from which the 
oxygen was removed with pyrogallol ^ was passed through a du- 
plicate series. The toxicity was determined with spores of C. Gos- 
sypii in closed-ring cells in the same manner as in Experiment i. 
Likewise, a similar series was arranged, using an aerated sus- 
pension of flowers of sulphur. The cultures were incubated at 
22° C. and the percentage of spore germination determined after 
18 hours. The results are recorded in table iv. 

The results of these tests prove conclusively that the toxic 
property of sulphur is due to an oxidation product and that finely 
divided sulphur is more readily oxidized at ordinary temper- 
atures than the ordinary sublimed sulphur. 

^One part pyrogaEol, 5 parts NaOH, and 30 parts HgO. 



1922] 



YOUNG THE TOXIC PROPERTY OF SULPHUR 

TABLE IV 

PERCENTAGE OP GERMINATION 
COLLETOTRICHUM GOSSYPII 



423 



Ph 


Hydrophobic colloidal sulphur 


Flowers of sulphur 




-02 


+ 02 


-02 


+ 02 


2.4 
3.2 
4.2 
5.4 
5.8 
6.4 



26 
42 
56 
60 
66 



18 

2 
13 
37 
62 




22 
51 
60 
68 
54 



18 
16 
10 
43 
56 



EXPERIMENT 7. THE INFLUENCE OF H2O ON THE TOXICITY 

OF SULPHUR 

The influence of water on this volatile compound was next 
studied. Dry colloidal sulphur was prepared by centrifuging hy- 
drophobic colloidal sulphur and the residue dried at room tem- 
perature. This was placed in the bottom of Van Tieghem cells. 
Spores of Sclerotinia cinerea were placed in sterile distilled water 
on sterile cover slips and inverted over the cell. The cell was not 
made air-tight, thereby not eliminating any other factor except 
water. Checks were arranged in which a suspension was used in- 
stead of the dry sulphur, other conditions being the same. All the 
cultures were placed in a moist chamber at room temperature. 
There resulted from this experiment no inhibition when dry sul- 
phur v/as used, while the suspension gave the same amount of in- 
hibition as reported in table iii. Oxygen and water are necessary 
factors in the formation of the toxic volatile compound of sulphur. 



Chemistry of Hydrophilic Colloidal Sulphur 

The results of all the foregoing experiments point towards 
hydrophilic colloidal sulphur as containing the toxic substance 
produced by the oxidation of the ordinary forms of sublimed 
and precipitated sulphur. It is as toxic in closed-ring cells 
where little oxygen is available as in open aerated cells. The 
other forms of sulphur tried are toxic only when oxygen is pres- 
ent. Hydrophilic colloidal sulphur is toxic at 21-22° C. to 
Botrytis cinerea, Macrosporium sarcinaeforme, Gloeosporium 
venetum, and Colletotrichum Gossypii, all of which are very re- 



[Vcl. 9 
424 ANNALS OF THE MISSOURI BOTANICAL GARDEN 

sistant and grow normally in a suspension of flowers of sulphur 
at temperatures below 25° C. Because of these facts it is logical 
to assume that the toxic property of sulphur is due to a compound 
formed by the oxidation of sulphur. Having eliminated the more 
common oxides and acids of sulphur it was thought that this 
toxic compound might be one or a mixture of the more complex 
polythionic acids. At any rate hydrophilic colloidal sulphur con- 
tains such an acid. The chemistry of hydrophilic colloidal sul- 
phur has been studied by a number of investigators. Bary ('20) 
studied Raffo's soluble sulphur (here termed hydrophilic colloidal 
sulphur), and came to the conclusion that the substance con- 
tained not only sulphur but polythionates. He thought the solu- 
tion was made stable by the presence of small amounts of electro- 
lytes. Freundlich and Scholz ('22) made a very extensive study 
of the so-called soluble sulphur and concluded that it was large- 
ly pentathionic acid. They base their conclusion on the following 
reactions which would take place if pentathionic acid were pre- 
sent. 

2NaOH + H2S5O6 = Na2S306 + S2 + 2H2O 
2 NacSsOs + 6NaOH =. Na2S203 + 4 Na2S03 -|- 3 H2O + 4S 
or 2Na2S506 + 6NaOH = 5Na2S203 + 3 H2O. 

By the aid of this reaction they were able to determine qualita- 
tively and quantitatively the pentathionic acid. The qualitative 
test was made by the addition of an alkali which precipitated out 
the sulphur in the form of a white turbid solution. They state 
that this test applies only to pentathionic acid, and to no other 
sulphur compound containing oxygen, such as any of the well- 
known acids or oxides. The quantitative test is made by treating 
the colloidal solution with normal NH4OH, forming ammonium 
thiosulphate and titrating this with 0.0 IN iodine solution. With 
hydrophobic colloidal sulphur these tests were negative. They 
designate colloidal sulphur in this form as S X and the form as- 
sociated with pentathionic acid as S |j. When S n is precipitated 
out of hydrophilic colloidal sulphur it probably becomes S X. 
Such a change also takes place when pentathionic acid is treated 
with H2S. 

According to these workers, sodium thiosulphate and sulphuric 
acid react as follows: 

Na2S203 + H2SO4 = H2S2O3 + Na2S04 
H2S2O3 = SO2 + 3S + H2O 



1922] 

YOUNG THE TOXIC PROPERTY OF SULPHUR 425 

By the action of remaining sulphuric acid, 

H2SO4 + 5H2S2O3 = 2H2S5O6 + 3 H2O 
The pentathionic acid then joins with sulphur (S p) and water 
to form the hydrophilic colloid of the following structure: 

SsOe H2 SsOe 
H2O 

The possibility of such a structure is based on the fact that a 
compound containing so many oxygen ions must necessarily have 
a great affinity for water. Moreover, a molecule containing so 
many sulphur atoms would, because of its residual valence, ac- 
count for its combining with other atoms of sulphur. This being 
true, pentathionic acid would have the property of combining 
between molecules of sulphur and water. In other words, it is 
an adsorptive medium for both these substances. A similar phe- 
nomenon is described and illustrated by Langmuir (17) in his 
studies of secondary valences in mixtures of fats and water. 

Having no such adsorption medium present in hydrophobic 
colloidal sulphur, the S A absorbs water and forms the grouping 
S A.' H2O, which is a typical suspension colloid, poorly hydrated 
and gradually settling out. 

The chemical nature of pentathionic acid has been very thor- 
oughly studied. It was discovered by Wackenroder ('46) in 
1845. He prepared the acid by passing H2S into a saturated 
solution of SO2, always keeping the excess of the latter. By 
calculations he arrived at the formula of H2S5O6. For quan- 
titative determinations he precipitated the acid with an alkali, 
in much the same way as reported by Freundlich and Scholz 
('22) for hydrophilic colloidal sulphur. He also found that salts 
precipitated it. 

After the discovery of this acid considerable controversy arose 
as to its existence in a pure state. Spring ('82) states that it is 
his opinion that the so-called pentathionic acid consists of a solu- 
tion of sulphur in tetrathionic acid and that salts obtained from 
this solution are simply tetrathionates containing admixed sul- 
phur. That this conclusion was partially correct was proved by 
Shaw ('83). He could produce pure pentathionic acid, but at 
times such an admixture as obtained by Spring would be obtained. 
A close relationship undoubtedly exists between pentathionic acid 



[Vol. 9 
426 ANNALS OF THE MISSOURI BOTANICAL GARDEN 

and sulphur. Shaw prepared his pentathionic acid by passing 
simultaneously hydrogen sulphide and sulphur dioxide into 3 
liters of distilled water for 32 hours, the sulphur dioxide being 
kept slightly in excess. This controversy was definitely settled 
by Debus ('88). His work is summed up by Mellor ('17) in the 
chapter on the compounds of sulphur and oxygen. 

The properties of pentathionic acid have been more recently 
studied by Raschig ('20) and Riesenfeld and Feld ('21). The 
latter state that the action of H2S and SO2 forms the hypothet- 
ical acid H2S3O4 as an intermediary product and that by its 
oxidation and reduction pentathionic acid is formed; they give 
the following reactions: 

2SO2 + H2S = H2S3O4 

H2S3O4 + SO2 = H2S4O6 ( rkx,;^„+;^^ u,r ar^ 

H2S3O4 + 6SO2 = 2H2O + H2S30e j ^^^^"*^^^ ^y ^^' 

H2S3O4 + 3H2S = 6S + 4H2O— Reduction by H2S 
5H2S3O4 = 3H2S5O6 + H2O— Polymerization 

They studied the action of acid and alkali and found that the 
acid was unstable in both conditions. 

It is therefore evident that the hydrophilic colloidal sulphur 
prepared according to the methods of Selmi ('52), Raffo ('08), 
Oden ('13), and others, is pentathionic acid. That this is an ox- 
idation product of sulphur seems a logical conclusion. The in- 
fluence of the hydrogen-ion concentration also points toward pen- 
tathionic acid as being the toxic factor in all of the preceding ex- 
periments. Flowers of sulphur, hydrophobic colloidal sulphur, 
and especially hydrophihc colloidal sulphur exhibited toxicity 
only at Ph 4.2-5.4, because of the fact that pentathionic acid 
is destroyed when in a solution of higher or lower hydrogen-ion 
concentration. 

To obtain further proof of the toxicity of pentathionic acid 
the hydrophilic colloidal sulphur was freed of this acid. The col- 
loidal sulphur was prepared by the following method, which is 
only a slight modification of the one used in previous experi- 
ments: Thirty cc. of a saturated solution of sodium thiosulphate 
were slowly added to 10 cc. of concentrated sulphuric acid. The 
mixture was warmed and filtered through glass wool. The fil- 
trate was then coagulated with sodium chloride and centrifuged. 
The coagulum was peptized in 100 cc. of distilled water and 
again centrifuged to remove insoluble sulphur. Coagulation, 



1922] 



YOUNG THE TOXIC PROPERTY OF SULPHUR 



427 



centrifuging, and peptizing were repeated 3 times, and the final 
coagulum peptized in 100 ce. of distilled water. The reaction of 
this peptized solution was Ph 4.2. Seventy-five cc. of this solution, 
for convenience designated No. 1, were treated with 25 cc. of 
normal ammonium hydroxide and let stand 24 hours, a white 
precipitate being formed. This was neutralized and centrifuged. 
The residue was suspended in 75 cc. of distilled water and desig- 
nated solution 2. The filtrate, No. 3, was again treated with 
25 cc. of normal ammonium hydroxide and left for 24 hours. A 
slight precipitate was formed. This was neutraUzed, the precip- 
itated sulphur centrifuged out and suspended in 10 cc. of water, 
and this last designated solution 4; and the filtrate, No. 5. 
Seventy-five cc. of the filtrate, No. 5, were again treated with 
25 cc. of normal ammonia and left for 24 hours. No precipitate 
formed. This was neutralized and called solution 6. 

Fifty cc. of solution 2 were treated with 25 cc. of normal 
ammonium hydroxide and kept for 24 hours. It was then 
neutralized and centrifuged. The residue was suspended in 
50 cc. of distilled water and designated solution 7. Twenty-five 
cc. of this solution were treated with 10 cc. of ammonia, allowing 
the usual interval, then again neutralized, centrifuged, and sus- 
pended in 25 cc. of water, constituting solution 8. A portion of 
each of these solutions was tested for pentathionic acid, with the 
result that Nos. 3, 5, 6, and 8 gave no positive test; Nos. 1, 2, 
and 7 gave positive tests, but 2 and 7 only a slight indication. 

These solutions were then tested in respect to toxicity in closed- 
ring cells, using the spores of Botrytis cinerea and Colletotrichum 
Gossypii. The cultures were placed at 22° C. for 24 hours, and 
the results, which are averages of duplicate cultures, are given in 
table V. 

TABLE V 
PERCENTAGE OF GERMINATION 



Organism 


No. of solution 


1 


2 


3 


4 


5 


6 


7 


8 


Ck. 


Botrytis cinerea 
Colletotrichum 
Gossypii 






10 

7 


50 
61 


41 

54 


55 
65 


55 
64 


26 
18 


54 
63 


53 
61 



The amount of killing was directly proportional to the amount 
of pentathionic acid present. No. 8 contained as much sulphur 
as No. 1 but was not toxic. 



[Vol. 9 
428 ANNALS OF THE MISSOURI BOTANICAL GARDEN 

Another experiment was conducted to ascertain if aerated 
flowers of sulphur produces pentathionic acid. Two lots of 50 
gms. each of flowers of sulphur were placed in wash bottles. 
The 2 bottles were placed in series each connected with wash 
bottles containing distilled water to collect any volatile water- 
soluble compound that might come over. Air was passed through 
one series and air deprived of oxygen through the other. Aeration 
was continued for 72 hours. At the end of this time H2S was 
passed into the distilled water wash bottles and permitted to 
stand for 12 hours. A slight precipitate was noted in the distilled 
water through which air containing oxygen had passed. The 
series without oxygen gave no precipitate. This afforded definite 
proof that pentathionic acid is an oxidation product of flowers of 
sulphur at ordinary temperatures. A concentrated solution of 
sodium chloride was added to the aerated sulphur suspensions, 
and centrif uged ; the residue was resuspended in water and again 
centrifuged. Hydrogen sulphide was then passed into the super- 
natant liquid. A precipitate developed only in the one in which 
oxygen was present. 

A similar series was arranged using precipitated sulphur con- 
taining no pentathionic acid. The distilled water containing 
the volatile soluble compound and the aerated suspension were 
tested for pentathionic acid. The former gave a slight precipitate 
with H2S after standing. The suspension gave a much heavier 
precipitate indicating that the pentathionic acid was adsorbed by 
the sulphur particle and was not easily driven off by slow aera- 
tion. Without oxygen there was no pentathionic acid produced. 

The precipitated sulphur was much more easily oxidized than 
the sublimed flowers of sulphur. 

Practical Applications 

Time has not permitted a more extensive study of this phase 
of the problem. It was necessary in the first place to determine 
the compound of sulphur that is toxic to fungi and to develop 
a material that would act as a fungicide over a sufficient period 
when sprayed on the plant. The fact that flowers of sulphur 
must be acted upon by some definite external physical factor has 
limited its use to only a small section of the country. It has been 
the aim in this work to develop, if possible, a sulphur compound 
that would exhibit fungicidal properties regardless of climatic 
factors and would for that reason have a wide usage over a large 



1922] 

YOUNG THE TOXIC PROPERTY OF SULPHUR 429 

part of the country. To accomplish this the material must yield 
readily the toxic compound, pentathionic acid. The reaction 
must be kept slightly acid (Ph 4.0-5.5), as this toxic compound 
is destroyed above or below this point. It must be readily oxidiz- 
able at ordinary temperatures. It should have great adhes- 
iveness ; it must not burn the leaves. 

Colloidal sulphur has all these properties when tested in the 
laboratory and greenhouse. It is almost impossible to wash it 
from the leaves of plants after it has dried. It is difficult to 
remove it with a strong stream of water. Certainly rain would 
have little effect upon it. 

That colloidal sulphur is readily oxidized has been demon- 
strated in the foregoing experiments. Kuhl ('21) states that col- 
loidal sulphur bears the same relation to atmospheric oxygen as 
phosphoric iron, the latter being self-inflammable. 

Methods for the preparation of colloidal sulphur mixtures for 
fungicidal use are being experimented upon. The hydrophilic 
colloidal sulphur prepared by the method given above is suitable 
as a spray. It did not burn the leaves of bean, potato, tobacco, 
rose, and geranium when sprayed on them. By the use of commer- 
cial materials this mixture is not too costly for practical purposes. 
Other methods for its preparation are being tried. 

The method for the preparation of hydrophobic colloidal sul- 
phur for trials in the greenhouse was as follows: One gallon 
of commercial or home-made lime sulphur was diluted with 5 gal- 
lons of water. Commercial phosphoric acid was added until the 
reaction was slightly acid. A milky precipitate of colloidal sul- 
phur was formed. The mixture was allowed to stand a day or 
two to remove excess H2S, and then applied. The advantage of 
phosphoric acid over other acids is that the calcium acid phos- 
phate formed maintains the proper reaction. This mixture diluted 
1 :5 with water prevented the germination of Botrytis cinerea and 
Colletotrichum Gossypii in aerated cultures. When sprayed on 
the plant this type of colloidal sulphur does not stick as well as 
hydrophilic colloidal sulphur but no doubt can be made just as 
effective a spray by the addition of soluble glue or other suitable 
spreaders. Any precipitated sulphur to which has been added 
calcium acid phosphate or another suitable compound for main- 
taining the slightly acid reaction should be an effective fungicide. 

With respect to increasing the value of flowers of sulphur as a 
spray the writer is not yet prepared to make a definite recom- 



[Vol. 9 
430 ANNALS OF THE MISSOURI BOTANICAL GARDEN 

mendation. However, the fact that this substance is slowly 
oxidized at ordinary temperatures leads to the possibility of its 
being used effectively when treated with compounds that will in- 
crease its oxidation. It will also be necessary to add to such a 
spray an adsorptive material to retain the pentathionic acid as it 
is produced. Many of the common spreaders now in use may 
do this. These possibilities are being investigated and will be 
reported later. 

Since the completion of the experimental part of this work 
there has come to my attention a number of colloidal sulphur 
preparations that have proved effective as a general spray. Ram- 
say and Cooke ('22) have prepared a colloidal sulphur that has 
been used effectively in Australia. They prepare their compound 
as follows: Ten gallons of home-made lime sulphur (26° Baume) 
are diluted with 25 gallons of water in a barrel of 50 gallons 
capacity. In a suitable vessel 6 pints of strong commercial sul- 
phuric acid are diluted with 9 parts of cold water and allowed to 
cool. The cold diluted sulphuric acid is then carefully added 
to the dilute lime sulphur in the barrel, a pint at a time, stirring 
well until the typical yellow color of the original lime sulphur 
disappears and until further addition of more acid produces no 
further precipitation of sulphur. The precipitated sulphur is 
allowed to settle for a day or two. Three pounds of cheap glue 
are dissolved in sufficient hot water to render the glue soluble and 
while still hot is stirred thoroughly into the sulphur. The glue aids 
in the keeping qualities of the colloidal sulphur. The mixture 
so prepared is diluted to 250 gallons (with water). This gives 
a spray containing approximately 5 pounds of precipitated sul- 
phur per 100 gallons. 

Thiele ('21) recommends the use of colloidal sulphur in the 
form of a liquid spray (not dust) for the control of mildews in 
Germany. He states that it is far more effective than the most 
finely ground sulphur powder. The colloidal mixtures adhere 
firmly to the plant and are not blown away by the wind or washed 
off by rains, as is the powder. Precipitated sulphur as a control 
for mildew and related fungi has been recommended by Lederle 
('22). He prepared this precipitated sulphur as follows: Solution 
I: 250 gms. of sodium hyposulphite are dissolved in % liter of 
hot water. Solution II: 250 gms. of sodium bisulphate are dis- 
solved in % liter of hot water. Solution III : 10 gms. of glue are 
dissolved in i/4 liter of hot water. Solution III is then stirred 



1922] 

YOUNG THE TOXIC PROPERTY OF SULPHUR 431 

while hot into solution I. After diluting solutions I and II each 
with 4 liters of water they are mixed and let stand for 3-18 hours 
when the mixture is ready for use. It is somewhat unstable 
and should be used within a few days, preferably the next morn- 
ing. 

Kuhl ('21) experimented with De Haen's colloidal soluble sul- 
phur ^ and found it to be very effective in controlling mildews 
and related diseases. He stated that the mixture was very ad- 
hesive and that it did not burn the leaves. He believed that the 
increased effectiveness of this type of sulphur over other sulphur 
sprays was due to its increased chemical activity. 

Barker and Wallace ('22) describe a new method for sulphur 
fumigation for the greenhouse. In previous studies they found 
that the fungicidal value of sulphur depended upon its being ap- 
plied as extremely finely divided particles. Their method is as 
follows: Air is passed through molten sulphur in a Campbell's 
"sulphur vaporiser," the temperature of the sulphur being kept 
just above the melting point and well below the ignition point. 
The melting point of sulphur is about 115° C. and its ignition 
point in the air is about 260° C. The most satisfactory tem- 
perature is around 170° C. Under these conditions an abun- 
dant cloud of sulphur in the particulate condition is produced. 
An improvement in the yield of particulate sulphur is effected 
if the current of air is passed into the molten sulphur through 
a perforated nozzle. By means of an attached delivery tube the 
particulate sulphur can be discharged in any given direction and 
on to any definite object. It can be used for general fumigation 
or for direct spraying. 

Another method for fumigation has been described by Vogt 
('21), and is as follows: Three-hundred gms. of pure roll sulphur 
(stick sulphur) contained in a small iron pan is liquefied and 
brought to the boiling point (448° C). There is heated at the 
same time in a circular copper boiler 400 gms. of water. The 
strongly superheated steam of the latter is forced under high 
pressure through the boiling sulphur which vaporizes it into 
small mist-like drops. These drops preserve their liquid form for 
several hours. They possess a high degree of adhesion not other- 
wise common to sulphur and do not burn the leaves. A few 
gms. of sulphur are enough to fill an average greenhouse with 
clouds of vapor which in a very short time covers all free surfaces. 

* Manufactured by De Haen at Seelze. 



[Vol. 9 
432 ANNALS OF THE MISSOURI BOTANICAL GARDEN 

A strong stream of water from the hose did not remove the sul- 
phur from panes of glass. The method is being perfected for 
open-air use. 

Conclusions 

1. Flowers of sulphur is not sufficiently toxic to inhibit the 
germination of spores of Botrytis cinerea, Colletotrichum Gos- 
sypii, Macrosporium sarcinaeforme, and Gloeosporium venetum 
in closed-ring cells at ordinary temperatures. Spores of Sclero- 
tinia cinerea and Phomopsis Sojae were inhibited from germin- 
ation. 

2. Finely ground flowers of sulphur was more toxic than the 
unground flowers under the same conditions but only at a hy- 
drogen-ion concentration of Ph 4.0-5.5. 

3. Methods for the preparation of hydrophilic and hydrophobic 
colloidal sulphur have been devised. 

4. Hydrophilic colloidal sulphur was extremely toxic to all the 
organisms used. 

5. Hydrophobic colloidal sulphur was slightly more toxic than 
the finely ground flowers of sulphur. 

6. The chemical and fungicidal properties of lime sulphur were 
studied. Before application lime sulphur contains 80.7 per cent 
sulphur as calcium sulphides, 19 per cent as calcium thiosulphate, 
and .03 per cent as sulphites and sulphates. After exposure to 
the air for a few hours as a spray the sulphides disappear and 
increasing amounts of sulphur are formed. The lasting effec- 
tiveness of the mixture is due to the precipitated sulphur which 
is about as toxic as hydrophobic colloidal sulphur. 

7. The toxic property of sulphur is not due to SO2, SO3 or H2S, 
or any of the common acids or oxides of sulphur, or to the sulphur 
particle. Germ tubes grew normally in a heavy suspension of pre- 
cipitated sulphur in closed-ring cells. 

8. The toxic property of sulphur is only exhibited when oxygen 
and water are present. 

9. By chemical analysis the toxic property of sulphur has been 
found to be pentathionic acid which is an oxidation compound 
formed from sulphur and water. 

10. Pentathionic acid is volatile and is an active adsorption 
compound. It is destroyed in acid and alkaline solutions. 



1922] 

YOUNG THE TOXIC PROPERTY OF SULPHUR 433 

11. Finely divided sulphur is more readily oxidized to penta- 
thionic acid at ordinary temperatures than is the flowers of 
sulphur. 

12. Finely divided sulphur has been used as a spray in Eng- 
land, Australia, and Germany, with excellent results. 

The writer takes pleasure in extending thanks to Dr. B. M. 
Duggar, who has directed the work, for many timely suggestions 
and criticisms; to Dr. George T. Moore, for the privileges and 
facihties of the Missouri Botanical Garden; and to the Crop 
Protection Institute for funds and materials. 

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Washington University 

Frederic A. Hall, A.M., Litt.D., L.H.D., LL.D., Bridge Chancellor 

I. The College (Skinker road and Lindell boulevard) 

George O. James, Ph.D., Dean 

II. The School of Engineering (Skinker road and Lindell boulevard) 
Walter E. McCourt, A.M., Dean 

III. The School of Architecture (Skinker road and Lindell boulevard) 
Walter E. McCourt, A.M., Dean 

iV. The School of Commerce and Finance 

(Skinker road and Lindell boulevard) 

William F. Gephart, Ph.D., Dean 
V. The Henry Shaw School of Botany 

(Shenandoah and Tower Grove avenues) 

George T. Moore, Ph.D., Engelmann Professor of 
Botany 

VI. The School of Graduate Studies (Skinker road and Lindell boulevard) 

Otto Heller, Ph.D., Chairman of the Board of 
Graduate Studies 

VII. The School of Law (Skinker road and Lindell boulevard) 
Richard L. Goode, A.M., LL.D., Dean 

VIII. The School of Medicine (Kingshighway and Euclid avenue) 

Nathaniel Allison, M.D., Dean 

IX. The School of Dentistry (Twenty-ninth and Locust streets) 
Walter Manny Bartlett, D.D.S., Dean 

X. The School of Fine Arts (Skinker road and Lindell boulevard) 
Edmund H. Wuerpel, Director 

XI. Division of University Extension (Skinker road and Lindell boulevard) 
Frederick W. Shipley, Ph.D., Director 



The following schools are also conducted under the charter of the 
University : 

Mary Institute — A Preparatory School for Girls 

(Waterman and Lake avenues) 

Edmund H. Sears, A.M., Principal 

The Training School for Nurses (600 S. Kingshighway) 
Helen Wood, A.B., Superintendent 



